应用双向热循环消减SELEX技术筛选胃癌血清核酸适配体

胃癌是发病率及死亡率均较高的消化道恶性肿瘤之一，严重威胁人类的生命健康。血清肿瘤标志物的检测对提高早期胃癌的检出率，改善胃癌的治疗有重要的意义。核酸适配体以其灵敏度高、靶向性强等优势显示出了较强的临床适用性。本研究以双向热循环消减指数富集式配基系统进化(systematic evolution of ligands by exponential enrichment，SELEX)技术为支持，纳米（琼脂）磁珠材料为载体，胃癌血清及正常人血清为筛选靶标，结合高通量测序技术建立了快速高效的核酸适配体筛选方法。经过19轮双向筛选，获得高重复率的胃癌血清特异性核酸适配体序列10条及正常人血清特异性核酸适配体序列8条。将这些序列分别混合并制成检测试剂A、B，结合实时荧光定量PCR（quantitative real-time PCR，qPCR）技术，对100份临床血清样本进行特异性验证。通过比较分析，建立了快速高效的胃癌血清检测技术。结果显示，核酸适配体G AP1与N AP1的二级结构类似于抗体的“Y”型，且呈茎环状。检测试剂A、B对胃癌及正常人血清的检出率分别为92%和88%。表明本技术可以较准确地筛选得到高特异性和强亲和力的核酸适配体，体现了核酸适配体作为新型肿瘤标志物在临床检测及治疗的应用潜力。

Screening of Nucleic Acid Aptamers in the Gastric Cancer Serum by Bidirectional Thermal Circulation Subtractive SELEX

Gastric cancer is one of the malignant tumors of the digestive tract with high morbidity and mortality, which seriously threatens human life and health. The detection of serum tumor markers is of great significance for improving the rate of early diagnosis of the gastric cancer and promoting the quality of gastric cancer therapy. Aptamer shows strong clinical applicability due to their high sensitivity and strong targetability. In this study, bidirectional thermal circulation subtractive systematic evolution of ligands by exponential enrichment (SELEX) technology was used to screen specific aptamer biomarkers in gastric cancer serum as compared with normal serum with the help of nano magnetic bead material and high-throughput sequencing technology. After 19 rounds of bidirectional screening, 10 gastric cancer serum specific and 8 normal serum-specific aptamer sequences with high repetition rates were selected for synthesis and preparation of detection reagent. Through comparative analysis，a quick and efficient serum detection technology was established with the help of qPCR technique. Sequence analysis results showed that the secondary structure of aptamers named G- AP1 and N-AP1 was stem-like and similar to the “Y” type of antibody. Statistical analysis showed that the positive detection rates of A and B for the two target sera were 92% and 88%, respectively. This technique could accurately screen aptamers with high specificity and strong affinity, which reflects the potential of aptamer as a new tumor biomarker in clinical detection and treatment.